Isolation and characterization of betaA3-crystallin associated proteinase from alpha-crystallin fraction of human lenses.
OBJECTIVE
The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses.


METHODS
An inactive, Arg-bond hydrolyzing proteinase in the alpha-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclusion Agarose A1.5 m chromatography, non-denaturing preparative gel-electrophoresis, and size-exclusion HPLC. The purified proteinase was characterized for the proteinase type, proteolysis of bovine recombinant gammaB-, gammaC-, and gammaD-crystallins, and its presence in three different protein fractions of human lenses (i.e., alpha-crystallin, beta(H)-crystallin, and membrane fractions).


RESULTS
An inactive, Arg-bond hydrolyzing proteinase present in the alpha-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl beta-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the alpha-crystallin fraction since it eluted at a lower molecular weight species than alpha-crystallin during size-exclusion Agarose A1.5 m chromatography. Following a three-step purification procedure, the enzyme showed three species between 22 kDa and 25 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The three protein bands were identified as betaA3-, betaB1-, and betaB2-crystallin by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitor studies revealed that the enzyme was a serine-type proteinase. Among the recombinant betaA3-, betaB1-, or betaB2-crystallins, only the betaA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of gammaC- and gammaD- crystallins, and the cleavage of gammaD-crystallin at M(1)-G(2), Q(54)-Y(55), M(70)-G(71), and Q(103)-M(104) bonds. Further, the enzyme was also present in three fractions of human lenses (alpha-crystallin, beta(H)-crystallin, and membrane fractions).


CONCLUSIONS
A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents. The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.
Srivastava
O P
OP
Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA. srivasta@uab.edu
Srivastava
K
K
Chaves
J M
JM
D000368:Aged; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003840:Deoxycholic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology*; D008875:Middle Aged; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000032:analysis; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013997:Time Factors; D038201:alpha-Crystallins / Q000378:metabolism*; D038205:beta-Crystallin A Chain / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D038201:alpha-Crystallins; D038205:beta-Crystallin A Chain; D003840:Deoxycholic Acid; D001120:Arginine; D010450:Endopeptidases